1. Field of the Invention
This invention is related in general to the field of light optical microscopy. In particular, it relates to a method and apparatus for conducting diagnostic testing of biological tissue with an array scanning microscope.
2. Description of the Related Art
Changes in the cellular structure of tissue are used to detect pathologic changes, to assess the progress of precancerous conditions, and to detect cancer. A tissue sample removed from a patient is typically sectioned and fixed to a slide for staining and microscopic examination by a pathologist. The morphology of the tissue (the visually perceptible structure and shape of features in the tissue) is analyzed to provide a qualitative assessment of its condition and to identify the presence of pathologic changes, such as may indicate progression towards a malignancy. For many decades, this visual procedure has been the diagnostic mainstay of pathology.
With the advent of computers and sophisticated digital imaging equipment, researchers have extended the realm of histopathology through the use of mechanized procedures for diagnostic and quantitative investigation. For example, U.S. Pat. No. 6,204,064 describes a method for measuring quantitatively the progression of a lesion toward malignancy by digitizing the images of clinical samples and analyzing nuclear chromatin texture features in the nuclei captured in the images. Numerical values are assigned to these features and compared to a monotonic progression curve previously established using the same criteria on known clinical samples ranging from normal to malignant tissue. Thus, the procedure provides a quantitative assessment of the condition of the tissue as well as a method for testing the efficacy of chemo-preventive drugs or therapeutic treatments.
In such mechanized procedures, histopathologic sections and/or cytologic preparations are imaged with a microscope and the images are digitized, stored, and analyzed for nuclear-placement patterns (histometry) or for the spatial and statistical distribution patterns of nuclear chromatin (karyometry). Karyometric assessment is always preceded by image segmentation, whereby each nucleus in an image is identified, outlined, isolated and stored as a separate image. For example, FIGS. 1 and 2 illustrate, respectively, the image of a histopathologic section and the enlarged image of a segmented nucleus and its chromatin pattern. As those skilled in the art readily understand, the nuclear chromatin pattern is an artifact of tissue fixation, but its spatial and statistical distributions are highly reproducible measures of the metabolic and functional state of cells. Thus, nuclear chromatin patterns have always been used in pathology to provide diagnostic clues. For example, the state of differentiation of the nucleus and its metabolic function may be reliably assessed based on a finding that nuclear chromatin is finely dispersed, coarsely aggregated, granular, clumped, or displaced toward the nuclear periphery.
Many chromatin texture features derived from the optical density of the tissue image have been identified as statistically significant for diagnostic purposes. Accordingly, after a sample is imaged and the image is digitized to provide an optical density value for each image pixel, the information is used in conventional manner first to identify and isolate each nucleus within the sample (image segmentation), and then to analyze chromatin patterns within each nucleus. The optical density recorded for each pixel is used to characterize chromatin features with statistical significance as parameters for identifying changes in the condition of the tissue. These features are then used much as alphabet letters can be used to identify features of a written text that are not readily perceptible by visual inspection. For example, the proportion of each letter appearing in a text, or the frequency of occurrence of certain letter diagrams, can be used to identify the language even though the text is not understood. Similarly, the spatial and statistical distribution of optical density in a nucleus can be used to detect chromatin patterns that are not visually perceptible. This notion provides a useful vehicle for achieving advantageous refinements in the detection of pathological change and of precancerous and cancerous lesions.
Optical density (OD) of a sample is defined in the art as the logarithm of the ratio of the light incident to the sample and the light transmitted through it. As used in microscopic imagery, optical density is usually expressed in terms of base-ten logarithmic values that range between zero and about 1.80 (because the accuracy of measurement limits near-zero transmission readings). An OD value of zero refers to full transmission, while an OD of 1.80 refers to transmission slightly greater than 1 percent. OD values are conventionally grouped into intervals of 0.10 OD units. For convenience, OD values may be multiplied by a factor of 100, so that computations can be carried out with integers (for example, an OD value of 1.0 is represented by 100, which corresponds to 10% light transmission).
As mentioned above, many features may be defined from the statistical and spatial distribution of nuclear chromatin. Global features are computed from the nucleus as a whole. For example, “total optical density” is defined as the sum of all pixel OD values in the nuclear area (i.e., the number of pixels within the outline of a nucleus). This feature is known to be related to the DNA ploidy of the nucleus, a measure of genetic instability and a diagnostic clue for progression toward a pre-malignant or malignant lesion. The variance of optical density within a nucleus is another example of global feature. Other features are local in nature, such as the frequency of occurrence of particular OD values within a certain interval, and have been identified in the art as indicative of tissue condition.
According to prior-art procedures, the chromatin features characterized using pixel OD values as described above have been reduced to number values representative of a quantitative measure of each feature and of a chromatin or nuclear “signature” representative of a set of features. These numeric values have then been used to provide pathologists with quantitative information available to complement their visual evaluation of tissue slides. For example, as generally described in U.S. Pat. No. 6,204,064, the information derived from the nuclear signature can be used advantageously as a quantitative measure of progression toward a lesion. That is, the physician is provided with information representative of a result formulated by the analytical algorithm built into the diagnostic system (e.g., a numerical value assigned to the nuclear signature calculated by the system and a resulting position in a progression curve). The physician is not provided with an image that has been information-enriched by the system and made available for visual inspection as an aid toward improved diagnostic evaluation. As a result, these prior-art mechanized procedures are often under-utilized by the profession. Moreover, the unique training and ability of pathologists to interpret visual imagery is not employed by the system.
Accordingly, there is still a need for a mechanized diagnostic system that provides information-enriched images of the tissue of interest in addition to the quantitative information generated by the analytical algorithm. Such a system would enable a pathologist to study histometric and karyometric features that are not visually detectable from the images acquired by the optical system but are rendered visible by the system's enhancing algorithms. Thus, significant chromatin features that might escape visual inspection become available for evaluation by the physician.
Obviously, the usefulness of any optical diagnostic technique is closely tied to the resolution of the imaging system because of the corresponding definition of chromatin patterns and the associated optical-density values. On the other hand, the image resolution of an optical objective is inversely related to its field of view. Therefore, any increase in resolution is accompanied by a corresponding loss of field of view, which greatly affects the system's capability for rapid imaging of useful-size tissue samples. Accordingly, in order to image a tissue sample with the degree of resolution required for cytopathological and histopathological testing, prior-art diagnostic imaging systems have relied on optics with high resolution and a small field of view, and on stitching techniques that yield a compound image from sequentially acquired images of adjacent portions of the tissue sample.
Typically, the field of view of a suitable microscope (such as a planapochromatic oil-immersion objective with NA 1.40 by Zeiss of Germany) is limited to about 200 microns. Related digital imaging systems enable sampling at six pixels per linear micron (36 pixels per square micron), as shown in FIG. 3, which results in several hundred to a few thousand pixels being recorded for each nucleus in the sample. Thus, tissue samples (up to about 50×25 mm in size) require multiple imaging steps and subsequent stitching of the resulting partial images of the sample. Because of the difficulty involved in aligning images of adjacent portions of the sample, the resulting composite images of overlapping features are often sufficiently misaligned to become reliable as a source of optical density information. The stage manipulation and the attendant time required to image an object under high magnification is particularly troublesome in pathology analysis because the diagnostic information in the tissue may be located in only a small portion of the tissue that is being imaged. In addition, the time for image acquisition is large enough to prevent analysis and/or data transmission in real time, which is becoming increasingly important in today's world of mechanization and shared information.
In view of the foregoing, there also remains a great need for a high-resolution imaging system capable of imaging an entire biological sample in a single scan. This invention is directed at a system that meets this requirement and provides information-enriched diagnostic images for direct visual analysis by practitioners.